Biocatalysts whole cell

Whole-cell biocatalysts are basically one-pot cascade reactions, with various enzymatic reactions being carried out concurrently within individual cells. Compared to purified enzymes, whole-cell biocatalysts are inexpensive and easily scalable and can be stably stored indefinitely. Certain enzymes are unstable and lose activity when purified from cells. Living cells also contain and regenerate otherwise expensive redox cofactors and, with metabolic engineering, can produce desired chemicals from inexpensive carbon and nitrogen sources [66, 67]. On the other hand, a major downside of using whole cells for biotransformations is the increased cost of product extrachon and purification from fermentahon broths. One also has to consider the 

Production of aromaticp-hydroxy-cinnamic acid and mandelic acid from glucose by whole ell biocatalysts. Enzymes marked ( ) are heterologously expressed. Depending on the desired product chemical, enzymes mediating competing pathways will be disrupted. 

Production of artemisinin and paclitaxel precursors by engineered whole-cell biocatalysts from glucose. Introduction of biosynthetic genes from Artemisia annua encoding the amorphadiene synthase and amorphadiene oxidase yielded microbial strains that produce arte-misinic acid. Artemisinic acid can be chemically converted into artemisinin, introduction of the Taxus genes encoding taxadiene synthase and taxadiene 5a-hydroxy-lase resulted in E. constrains that produce key paclitaxel intermediates. The biosynthetic pathway for paclitaxel has not been fully elucidated. 

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In the present work, for detail kinetic studies, we compared biocatalytic reaction kinetics for four types of whole cell biocatalyst systems whole cells with periplasmic-secreting OPH under trc or T7 promoters and whole cells with cytoplasmic-expressing OPH imder trc or T7 promoters. 

Lactobacillus kefir was also employed as the whole-cell biocatalyst for the asymmetric reduction of ethyl 4-chloroacetoacetate to ethyl (.S )-4-chloro-3-hydroxybutanoate, the chiral... 

Recombinant Whole-Cell Biocatalysts Overexpressing Catalytic Enzymes... 

Figure 7.9 Reduction of a-chloro-3 -chloroacetophenone catalyzed by a whole-cell biocatalyst and the corresponding purified enzyme...

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

Carballeira, J.D., Alvarez, E., Campillo,M,etal. (2004)DiplogelasinosporagrovesiilNll 171018, anew whole cell biocatalyst for the stereoselective reduction of ketones. Tetrahedron Asymmetry, 15 (6), 951-962. 

A prochiral bis(cyanomethyl) sulfoxide was converted into the corresponding mono-acid with enantiomeric excesses as high as 99% using a nitrilase-NHase biocatalyst. The whole-cell biocatalyst Rhodococcus erythropolis NCIMB 11540 and a series of commercially available nitrilases NIT-101 to NIT-107 were evaluated in this study. As outlined in Figure 8.18, the prochiral sulfoxide may be transformed into five different products (plus enantiomeric isoforms), of which, three are chiral (A, B, and C) and two achiral (D and E). Only products A, B, and E were observed with the biocatalysts employed in this investigation. Both enantiomerically enriched forms of both A and C could be obtained with one of the catalysts used. The best selectivities are as follows (S)-A 99% ee, (R)-A 33% ee, (S)-C 66% ee, and (R)-C 99% ee, using NIT-104, NIT-103, NIT-108, and NIT-107 respectively. Each of these catalysts produced more... 

Wu, P.-H., Nair, G.R., Chu, I.-M. and Wu, W.-T. (2008) High cell density cultivation of Escherichia coli with surface anchored transglucosidase for use as whole-cell biocatalyst for a-arbutin synthesis. Journal of Industrial... 

The invented biocatalysts based on R. rhodochrous strain ATCC No. 53968 and on B. sphaericus strain ATCC No. 53969, were protected not only as whole cell biocatalysts, but also their derivatives. Biocatalyst definition includes in addition to whole cells cell membranes, cell extracts and enzymes from those microorganisms. It should be noted that the first six patents are actually sets of similar patents with the first one providing coverage in Europe and the second one in US. This strategy involves coverage in US as well as Europe (the total number of patents is higher than the number of inventions) however, the allowed claims in US were always smaller than that allowed in Europe. IGT s last patent (May 1996) was filed in July 1994, when they already had begun... 

In a patent on biological desulfurization [100] of petroleum/coal, only the use of whole cell biocatalysts was claimed. The biocatalysts included microorganisms belonging to the genus Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus or Corynebacterium. The desulfurization pathway (sulfur-specific vs. destructive) was not specified. The Japanese patents No. JP2071936C and JP7103379B seem to be equivalent patents. 

Whole-cell biocatalysts, organic solvents and, 16 412-413 Whole cells, 3 669-671 Whole-cell systems ionic liquids in, 26 897 Whole cluster pressing in white wine, 26 311 Whole-wheat flour, 26 279, 283 Whole yeast vaccines, 26 488 Who needs it concept, 24 190 Wicking limit, heat pipe, 13 230 Wicks... 

To search for an appropriate whole-cell biocatalyst, it is necessary to identify an organism that contains large amounts of the desired enzyme. Equally important, the organism should not contain related pathway enzymes that modify or destroy the product synthesized by the desired enzyme. 

SYNTHESIS OF CATECHOLS BY A RECOMBINANT WHOLE CELL BIOCATALYST... 

Figure 15.12 Two-liquid-phase-based biooxidation of styrene to styrene oxide with a recombinant whole-cell biocatalyst.

LIKELY COSTS OF LARGE SCALE HYDROXYLATION OR EPOXIDATION PROCESSES WITH WHOLE CELL BIOCATALYSTS... 

Bacillus subtilis, engineered to overproduce epoxide hydrolase, was used as a whole-cell biocatalyst to resolve racemic 1-benzyloxymethyl-1-methyloxirane with high (5)-selectivity. The remaining (/ )-epoxide was subsequently ring opened in situ, with inversion of stereochemistry, to obtain highly enantiomerically enriched (/ )-3-benzyloxy-2-methylpropane-l,2-diol in greater than 50 % theoretical yield (Figure 5.2). 

Hsu et have cloned two enzymes from Deimcoccus radiodurans for overexpression in E. coli in order to carry out a dynamic kinetic resolution to obtain L-homophenylalanine, frequently required for pharmaceutical synthesis. The starting material is the racemic mixture of A acetylated homophenylalanine, and the two enzymes are an amino acid A -acylase, which specifically removes the acetyl group from the L-enantiomer, and a racemase, which interconverts the D- and L-forms of the A acyl amino acids. The resolution was carried out successfully using whole-cell biocatalysts, with the two enzymes either expressed in separate E. coli strains or coexpressed in the same cells. 

The microbial sources of penicillin amidases/acylases required for side-chain removal were found and were quickly commercialised as whole-cell biocatalysts. 

The commercial availability of enzymes or whole cell biocatalysts for a desired biotransformation is freqnently a limiting factor for commercial application of biocatalysts. Enzymes that are cheaply available are typically used in detergents, processing of food, feed and textiles, as well as in waste management applications. Most of these are hydrolytic enzymes, bnt also isomerases (e.g. glucose isomerase) and oxidorednctases are used on indnstrial scale (Table 5.1). 

Whole cell biocatalysts are more difficult to obtain and apply than enzymes. Although numerous strain collections exist that can supply strains with known biotransformation activities (see paragraph 5.2.1), one has to be able to cultivate the micro-organisms or perform expensive toll-fermentations elsewhere to obtain enough... 

Biocatalysts based on hydrolases (E.C. class 3, Table 5.2) ate mostly used as (purified) enzymes since they are cofactor independent, since these preparations are commercially available and because a number of hydrolases can be applied in organic solvents. Oxidoreductases (E.C. class 1) however, are relatively complex enzymes, which require cofactors and frequently consist of more than one protein component. Thus, despite the fact that efficient cofactor regeneration systems for NADH based on formate dehydrogenase (FDH) have been developed (Bradshaw et al, 1992 Chenault Whitesides, 1987 Wandrey Bossow, 1986, chapter 10) and that also an NADPH dependent FDH has been isolated (Klyushnichenko, Tishkov Kula, 1997), these enzymes are still mostly used as whole-cell biocatalysts. 

Physiological optimization of enzyme synthesis by variation of the culture parameters is usually required to enhance the catalytic activity of whole-cell biocatalysts to such a level that it can be apphed in a biocatalytic process. In addition, physiological conditions can influence the selectivity of the reaction, since enzymes with opposite selectivities can be differentially expressed. In some cases, genetic engineering is required to obtain biocatalysts with a desired selectivity that does not consume the product of choice (see 5.3.5). Alternatively, one may choose to isolate the desired activity from the culture in order to use the biocatalyst in an enzyme reactor. 

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